富氢水对创伤性脑损伤大鼠炎性因子及线粒体损伤的影响

Effect of hydrogen-rich water on the chondriosome damage and cytokines in brain tissue of rats with traumatic brain injury

目的观察富氢水对创伤性脑损伤（TBI）大鼠创伤周围脑组织线粒体损伤及炎症反应的影响。方法将54只健康雄性SD大鼠按随机数字表法分为假手术组（Sham组）、创伤组（TBI组）及创伤＋富氢水组（TBI＋HW组），每组分为伤后1、3、7 d 3个亚组，每个亚组6只。采用改良Feency自由落体撞击法制备TBI大鼠模型；Sham组开颅窗后不予以颅脑撞击。TBI＋HW组于制模后立即腹腔注射富氢水5 mL/kg，每日1次，直至处死；Sham组和TBI组均给予等量生理盐水。各组于伤后相应时间点进行神经损伤评分（NSS）后断头处死大鼠，取创伤边缘3 mm内脑组织，采用酶联免疫吸附试验（ELISA）检测炎性因子水平，用蛋白质免疫印迹试验（Western Blot）检测凋亡相关蛋白Bax、Bcl-2蛋白表达，用荧光酶标法检测线粒体活性氧（ROS）、线粒体膜电位（MMP）和线粒体膜通透性转换孔（MPTP）。结果TBI组和TBI＋HW组大鼠伤后出现明显神经功能损害，但TBI＋HW组伤后3 d和7 d神经功能损害明显轻于TBI组〔NSS（分）：9.67±0.82比11.17±1.17，6.83±0.75比8.50±1.04，均P〈0.05〕。与Sham组比较，TBI组和TBI＋HW组各时间点脑组织肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、ROS、Bax蛋白表达均明显升高，并于1 d达峰值后逐渐下降；各时间点MMP、MPTP及Bcl-2蛋白表达均明显下降，并于1 d达谷值后逐渐上升。与TBI组比较，TBI＋HW组1 d起TNF-α、IL-1β、ROS、Bax蛋白表达即明显降低〔TNF-α（ng/L）：54.14±1.11比81.49±2.76，IL-1β（ng/L）：74.53±1.75比119.44±3.56，ROS（RFU）：92.30±2.46比121.33±6.57，Bax蛋白表达：0.89±0.01比1.10±0.01，均P〈0.01〕，MMP、MPTP和Bcl-2蛋白表达即明显升高〔MMP（RFU）：99.28±3.97比74.72±3.00，MPTP（RFU）：188.82±4.44比160.01±2.04，Bcl-2蛋白表达：0.52±0.02比0.30±0.02，均P〈0.01〕。结论大鼠TBI早期即有脑组织炎性因子表达增高及线粒体损伤；早期腹腔注射富氢液可减少线粒体损伤和炎性因子释放，减轻TBI后神经细胞凋亡，从而发挥脑保护作用。

ObjectiveTo observe the effect of hydrogen-rich water on the chondriosome damage and cytokines change in brain tissue of rats with traumatic brain injury （TBI）.MethodsFifty-four health male Sprague-Dawley （SD） rats were divided into three groups by random number table： sham group, trauma group （TBI group）, and trauma＋hydrogen-rich water group （TBI＋HW group）, the rats in each group were subdivided into 1, 3 and 7 days subgroups according to the time points after trauma, with 6 rats in each subgroup. The TBI model was reproduced by using a modified Feency method for free fall impact, and the rats in sham group were not given brain impact after craniotomy. The rats in TBI＋HW group were given intraperitoneal injection of hydrogen-rich water （5 mL/kg） after TBI model reproduction, and then once a day until being sacrificed; and the rats in sham group and TBI group were given the same amount of normal saline. The neurological severity scores （NSS） for neurologic deficits were calculated at corresponding time points, and then the rats were sacrificed to harvest brain tissue at 3 mm around lesion boundary. The cytokines including tumor necrosis factor-α （TNF-α） and interleukin-1β（IL-1β） were determined by enzyme linked immunosorbent assay （ELISA）; the protein expressions of Bax, Bcl-2 were determined by Western Blot; the RFU of reactive oxygen species （ROS）, mitochondrial membrane potential （MMP） and mitochondrial membrane permeability （MPTP） were determined by fluorescence and enzyme sign method.ResultsTBI and TBI＋HW groups appeared obvious neurologic damage after injury in rats. NSS scores in TBI and TBI＋HW groups showed a decreased tendency with time prolongation after TBI. NSS scores in TBI＋HW group at 3 days and 7 days were significantly lower than those of TBI group （NSS score： 9.67±0.82 vs. 11.17±1.17, 6.83±0.75 vs. 8.50±1.04, both P 〈 0.05）. Compared with sham group, the expressions of TNF-α, IL-1β, RFU of ROS in chondriosome, protein expression of Bax in brain tissue in TBI group and TBI＋HW group were significantly increased, peaked at 1 day, then they gradually declined. Each time point of RFU of MMP, MPTP in chondriosome and protein expression of Bcl-2 were significantly decreased, and gradually increased after one-day valley value. Compared with TBI group, the expressions of TNF-α, IL-1β, RFU of ROS in chondriosome and protein expression of Bax in brain tissue were all declined at corresponding time points [TNF-α（ng/L）： 54.14±1.11 vs. 81.49±2.76, IL-1β（ng/L）： 74.53±1.75 vs. 119.44±3.56, ROS （RFU）： 92.30±2.46 vs. 121.33±6.57, Bax： 0.89±0.01 vs. 1.10±0.01, all P 〈 0.01]; RFU of MMP, MPTP in chondriosome and the protein expression of Bcl-2 were all increased at corresponding time points [MMP （RFU）： 99.28±3.97 vs. 74.72±3.00, MPTP （RFU）： 188.82±4.44 vs. 160.01±2.04, Bcl-2： 0.52±0.02 vs. 0.30±0.02, all P 〈 0.01].ConclusionsThe high expressions of cytokines and chondriosome damage were involved in the early TBI. Early treatment with an intraperitoneally injection of hydrogen-rich water can reduce chondriosome damage and inflammation factor release, reduce the nerve cell apoptosis after TBI, and protect brain function.